Unfolded protein response in Gaucher disease: from human to Drosophila

Abstract

Background: In Gaucher disease (GD), resulting from mutations in the GBA gene, mutant β-glucocerebrosidase (GCase) molecules are recognized as misfolded in the endoplasmic reticulum (ER). They are retrotranslocated to the cytoplasm, where they are ubiquitinated and undergo proteasomal degradation in a process known as the ER Associated Degradation (ERAD). We have shown in the past that the degree of ERAD of mutant GCase correlates with GD severity.Persistent presence of mutant, misfolded protein molecules in the ER leads to ER stress and evokes the unfolded protein response (UPR).

Methods: We investigated the presence of UPR in several GD models, using molecular and behavioral assays.

Results: Our results show the existence of UPR in skin fibroblasts from GD patients and carriers of GD mutations. We could recapitulate UPR in two different Drosophila models for carriers of GD mutations: flies heterozygous for the endogenous mutant GBA orthologs and flies expressing the human N370S or L444P mutant GCase variants. We encountered early death in both fly models, indicating the deleterious effect of mutant GCase during development. The double heterozygous flies, and the transgenic flies, expressing mutant GCase in dopaminergic/serotonergic cells developed locomotion deficit.

Conclusion: Our results strongly suggest that mutant GCase induces the UPR in GD patients as well as in carriers of GD mutations and leads to development of locomotion deficit in flies heterozygous for GD mutations.

Figure 1

Elevation in CHOP and BiP levels in GD derived cells. A. RNA was isolated from different GD derived skin fibroblasts, and the corresponding cDNA was used for quantitative RT-PCR with primers specific for human BiP or CHOP. GAPDH was used as a normalizing control. Two different normal cell lines were used as control. Dark box: CHOP; Light box: BiP. B, C. Protein lysates were prepared from different GD derived skin fibroblasts and subjected to western blotting. The corresponding blots were interacted with anti BiP (B) and anti CHOP (C) antibodies. As a loading control, the blots were interacted with anti-tubulin antibody. For each protein there are two blots, each with a normal control. This is due to the fact that cell lysates were prepared at different times, depending on the growth rate of the cell lines and, therefore, ran on different gels. The genotypes for B and C are shown in D. D. The blots were quantified as explained. The amount of BiP and CHOP was divided by that of tubulin in the same lane, and the values obtained for normal cells were considered 1. The results are the mean (minus plus standard error) of three different experiments. Significance: * < 0.05; ** < 0.01.

Figure 2

Elevation in Xbp1 splicing and eIF2α phosphorylation in GD derived cells. A. Scheme showing the two Xbp1 RNA variants, the spliced and the non-spliced forms. B. RNA was isolated from different GD derived skin fibroblasts, and the cDNA prepared from it was used for RT-PCR, with primers specific for the spliced form of human Xbp1. GAPDH was used as a normalizing control. C. The results (three different experiments) were quantified and the amount of Xbp1 was divided by that of GAPDH in the same lane. The values obtained for normal cells were considered 1. D. Protein lysates were prepared from different GD derived skin fibroblasts and subjected to western blotting. The corresponding blots were interacted with anti phosphorylated eIF2α (p-eIF2α) and as a loading control, with anti eIF2α antibodies. E. p-eIF2α amount was divided by that of eIF2α in the same lane, and the values obtained for normal cells were considered 1. The results are the mean (minus plus standard error) of three different experiments. Significance: * < 0.05; ** < 0.01. The genotypes for B and D are shown in C and E, respectively.

Figure 3

Activation of UPR in carriers of GD mutations. A. RNA was isolated from skin fibroblasts that originated from carriers of GD mutations, and the corresponding cDNA was used for quantitative RT-PCR with primers specific for human BiP or CHOP. GAPDH was used as a normalizing control. B. cDNA, prepared as in (A), was subjected to RT-PCR, with primers specific for the spliced form of human Xbp1. GAPDH was used as a normalizing control. C. The results (three different experiments) were quantified as explained in the legend to Figure 2 and the values obtained for normal cells were considered 1. D. Protein lysates, prepared from the above-mentioned cells, were subjected to western blotting and interaction with anti phosphorylated eIF2α antibodies (p-eIF2α). As a loading control, the blots were interacted with anti eIF2α antibodies. E. The results (three different experiments) were quantified as explained in the legend to Figure 3B, and the values obtained for normal cells were considered 1. There are two blots, each with a normal control. This is due to the fact that cell lysates were prepared at different times, depending on the growth rate of the cell lines and, therefore, ran on different gels. Significance: * < 0.05; ** < 0.01; *** < 0.005. The genotypes for B and D are shown in C and E, respectively.

Figure 4

UPR activation in Drosophila carriers of mutant GBA orthologs. A. GCase activity was tested in protein lysates prepared from either adult wild type (Canton S, WT) flies or from males and females of double heterozygous flies (Mi{ET1}CG31148,CG31414/CG31148,Mi{ET1}CG31414) using 4-MUG as a substrate. Presented are results of three experiments. B. RNA was isolated from double heterozygous flies, and the cDNA prepared from it was subjected to quantitative RT-PCR with primers specific for Drosophila Hsc-70-3 or for the spliced form of Drosophila Xbp1. The results (three different experiments) were quantified as explained in the legend to Figure 1A, and the values obtained for WT flies were considered 1. RP49 was used as a normalizing control. Dark box: WT flies; light box: double heterozygous flies. C. Protein lysates from double heterozygous flies (Double hets.) were subjected to western blotting and interaction with anti phosphorylated eIF2α antibodies (p-eIF2α). D. The results (three different experiments) were quantified as explained in the legend to Figure 2E and the values obtained for WT flies were considered 1. Significance: * < 0.05; ** < 0.01. E. The number of larvae, (L3,) that survived to the pupal stage, and the number of adults that eclosed was counted. The number of larvae taken for each experiment was 90.

Figure 5

Activation of UPR in Drosophila expressing the human N370S or L444P mutant variants. A. Protein lysates were prepared from 10 flies expressing the human UAS-mycHisWTGCase, human UAS-mycHisN370SGCase, or human UAS-mycHisL444PGCase, driven by Da^Gal4. Samples, containing 100 μg of protein, were subjected to overnight endo-H digestion, after which they were electrophoresed through SDS-PAGE and the corresponding blot was interacted with anti GCase and anti actin antibodies. Total GCase amount was divided by that of actin at the same lane and normalized to WT GCase, which was considered 100. The endo-H sensitive fraction before and after endo-H treatment, and the endo-H resistant fraction were labeled for convenience, as follows: endo-H sensitive before treatment: * [Endo-Hs(−)]; endo-H sensitive after treatment: # [Endo-Hs(+)]; endo-H resistant: • [Endo-Hr(+)]. B. RNA was isolated from the above-mentioned flies, and the cDNA prepared from it was subjected to quantitative RT-PCR with the appropriate primers. RP49 was used as a normalizing control. The results (three different experiments) were quantified as explained in the legend to Figure 3B and the values obtained for flies expressing normal human GCase were considered 1. Significance: * < 0.05; ** < 0.01. C. Protein lysates, prepared from the above–mentioned flies, were subjected to western blotting and interaction with anti phosphorylated eIF2α antibodies (p-eIF2α). As a loading control, the blot was interacted with anti eIF2α antibodies. D. The results (three different experiments) were quantified as explained in the legend to Figure 3B and the values obtained for wild type flies were considered 1. E. The number of larvae, (L3), that survived to the pupal stage, and the number of pupae that eclosed was counted. The number of larvae taken for each experiment was 90.

Figure 6

Locomotion test in flies. A. Five vials, each containing flies expressing the human UAS-mycHiswtGCase, human UAS-mycHisN370SGCase, or human UAS-mycHisL444P GCase, driven by the DdcGal4 driver, were analyzed for locomotion behavior. B. The same experiment as described in A was performed using the WT and double heterozygous flies.